Project summary. The innate immune system plays an essential role in protecting host tissue function by eradicating pathogens and other foreign elements, promoting proper wound healing, and maintaining tissue homeostasis. However, dysregulated innate immune system activation resulting from aberrant self-directed immune responses can lead to states of chronic local inflammation that induce tissue damage or dysfunction. Chronic inflammatory diseases are often incurable, and therefore the current standard of care involves managing pain or fever via drugs that inhibit pro-inflammatory cytokines or their receptors. Although effective, these treatment modalities can suppress immune system function and render patients susceptible to opportunistic infections. In contrast, natural resolution of inflammation is mediated in part by enzymes anchored to the cell membrane that convert immunostimulatory signals to an inactive, or in some cases immunosuppressive, form. For example, extracellular ATP (eATP) released by one?s own damaged or dying cells acts as a ?danger signal? that activates inflammation, and eATP immunostimulatory activity is locally regulated by membrane-anchored enzymes that dephosphorylate ATP to adenosine (Ado), an immunosuppressive signal. Inspired by these observations, an ATP dephosphorylating enzyme, apyrase, is currently investigated as an immunotherapeutic biologic, and has demonstrated efficacy for suppressing inflammation in pre-clinical models. However, clinical efficacy of soluble apyrase delivered via parenteral routes is likely to be hindered by the short effective half-life typical of biologic drugs. To address this limitation, the proposed research program will develop biomaterials with integrated ATP dephosphorylating enzymes as immunotherapeutics that can be locally delivered to specific tissue sites to suppress aberrant inflammation. Toward this end, the proposed research program will create hydrated polymeric gels (i.e. ?hydrogels?) of self- assembled peptide nanofibers with integrated enzymes that dephosphorylate ATP to Ado. Specifically, we will adapt our established platform, Co-Assembly Tags based on CHarge complementarity (?CATCH?), to create hydrogels harboring Adenosine Synthase A (AdsA), an enzyme that dephosphorylates ATP to Ado. Through this grant, we will (i) optimize CATCH-AdsA hydrogel enzymatic activity through material redesign, (ii) assess CATCH-AdsA hydrogel efficacy for immunomodulation using in vitro and in vivo models, and (iii) establish a pre-clinical safety profile for these biomaterials by assessing host innate and adaptive immune responses to CATCH hydrogels and their individual components. Success of the proposed research will lead to new biomaterials that can locally suppress inflammation via presentation of an immunomodulatory enzyme, which will provide the basis for future efforts to develop new immunotherapeutics to resolve chronic inflammation. More generally, a biomaterial platform with interchangeable integrated enzyme components is likely to enable new opportunities to harness natural enzymatic mechanisms to treat various immune-related pathologies.